Two distinct ferredoxins are essential for nitrogen fixation by the iron nitrogenase in Rhodobacter capsulatus

ABSTRACT Nitrogenases are the only enzymes able to fix gaseous nitrogen into bioavailable ammonia and hence are essential for sustaining life. Catalysis by nitrogenases requires both a large amount of ATP and electrons donated by strongly reducing ferredoxins or flavodoxins. Our knowledge about the mechanisms of electron transfer to nitrogenase enzymes is limited: The electron transport to the iron (Fe)-nitrogenase has hardly been investigated. Here, we characterized the electron transfer pathway to the Fe-nitrogenase in Rhodobacter capsulatus via proteome analyses, genetic deletions, complementation studies, and phylogenetics. Proteome analyses revealed an upregulation of four ferredoxins under nitrogen-fixing conditions reliant on the Fe-nitrogenase in a molybdenum nitrogenase knockout strain, compared to non-nitrogen-fixing conditions. Based on these findings, R. capsulatus strains with deletions of ferredoxin (fdx) and flavodoxin (fld, nifF) genes were constructed to investigate their roles in nitrogen fixation by the Fe-nitrogenase. R. capsulatus deletion strains were characterized by monitoring diazotrophic growth and Fe-nitrogenase activity in vivo. Only deletions of fdxC or fdxN resulted in slower growth and reduced Fe-nitrogenase activity, whereas the double deletion of both fdxC and fdxN abolished diazotrophic growth. Differences in the proteomes of ∆fdxC and ∆fdxN strains, in conjunction with differing plasmid complementation behaviors of fdxC and fdxN, indicate that the two Fds likely possess different roles and functions. These findings will guide future engineering of the electron transport systems to nitrogenase enzymes, with the aim of increased electron flux and product formation. IMPORTANCE Nitrogenases are essential for biological nitrogen fixation, converting atmospheric nitrogen gas to bioavailable ammonia. The production of ammonia by diazotrophic organisms, harboring nitrogenases, is essential for sustaining plant growth. Hence, there is a large scientific interest in understanding the cellular mechanisms for nitrogen fixation via nitrogenases. Nitrogenases rely on highly reduced electrons to power catalysis, although we lack knowledge as to which proteins shuttle the electrons to nitrogenases within cells. Here, we characterized the electron transport to the iron (Fe)-nitrogenase in the model diazotroph Rhodobacter capsulatus, showing that two distinct ferredoxins are very important for nitrogen fixation despite having different redox centers. In addition, our research expands upon the debate on whether ferredoxins have functional redundancy or perform distinct roles within cells. Here, we observe that both essential ferredoxins likely have distinct roles based on differential proteome shifts of deletion strains and different complementation behaviors.


Supplementary methods:
Proteome analysis R. capsulatus strains were cultured anaerobically until a total OD660 of 3 was achieved.Cell samples were prepared by three centrifugation steps and two washing steps with phosphate buffer (3.6 g Na2HPO4 × 2 H2O and 2.6 g KH2PO4 per litre distilled H2O).For protein extraction frozen cell pellets were re-suspended in 2% sodium lauroyl sarcosinate (SLS) and heated for 15 min at 90°C.Proteins were reduced with 5 mM Tris(2-carboxyethyl) phosphine (Thermo Fischer Scientific) at 90°C for 15 min and alkylated using 10 mM iodoacetamid (Sigma Aldrich) at 20°C for 30 min in the dark.Proteins were precipitated with a 6-fold excess of ice cold acetone, followed by two methanol washing steps.Dried proteins were reconstituted in 0.2 % SLS and the amount of proteins was determined by bicinchoninic acid protein assay (Thermo Scientific).For tryptic digestion 50 µg protein was incubated in 0.5% SLS and 1 µg of trypsin (Serva) at 30°C overnight.
After digestion, SLS was precipitated by adding a final concentration of 1.5% trifluoroacetic acid (TFA, Thermo Fischer Scientific).Peptides were desalted by using C18 solid phase extraction cartridges (Macherey-Nagel).Cartridges were prepared by adding acetonitrile (ACN), followed by equilibration with 0.1% TFA.Peptides were loaded on equilibrated cartridges, washed with 5% ACN and 0.1% TFA containing buffer and finally eluted with 50% ACN and 0.1% TFA.
Peptides were dried and reconstituted in 0.1% trifluoroacetic acid and then analyzed using liquidchromatography-mass spectrometry carried out on a Exploris 480 instrument connected to an Ultimate 3000 RSLC nano and a nanospray flex ion source (all Thermo Scientific).Peptide separation was performed on a reverse phase HPLC column (75 μm x 42 cm) packed in-house with C18 resin (2.4 μm; Dr. Maisch).The following separating gradient was used: 94% solvent A (0.15% formic acid) and 6% solvent B (99.85% acetonitrile, 0.15% formic acid) to 25% solvent B over 95 minutes at a flow rate of 300 nl/min, and an additional increase of solvent B to 35% for 25min.MS raw data was acquired in data independent acquisition mode with a method adopted from Bekker-Jensen et al. [1] .In short, Spray voltage were set to 2.3 kV, funnel RF level at 40, and heated capillary temperature at 275 °C.For DIA experiments full MS resolutions were set to 120.000 at m/z 200 and full MS, AGC (Automatic Gain Control) target was 300% with an IT of 50 ms.Mass range was set to 350-1400.AGC target value for fragment spectra was set at 3000%.45 windows of 14 Da were used with an overlap of 1 Da.Resolution was set to 15,000 and IT to 22 ms.Stepped HCD collision energy of 25, 27.5, 30 % was used.MS1 data was acquired in profile, MS2 DIA data in centroid mode.Analysis of DIA data was performed using DIA-NN version 1.8 using a UniProt protein database from Rhodobacter capsulatus [2] .Full tryptic digest was allowed with two missed cleavage sites, and oxidized methionines and carbamidomethylated cysteine.Match between runs and remove likely interferences were enabled.The neural network classifier was set to the single-pass mode, and protein inference was based on genes.Quantification strategy was set to any LC (high accuracy).Cross-run normalisation was set to RT-dependent.Library generation was set to smart profiling.DIA-NN outputs were further evaluated using the SafeQuant and script modified to process DIA-NN outputs [3,4] .The SafeQuant script was executed on the "report.tsv"file from DIA-NN analysis to sum precursor intensities to represent protein intensities.The peptide-to-protein assignment was done in SafeQuant with redundant peptide assignment following the Occam´s razor approach.Median protein intensity normalization was performed followed by imputation of missing values using a normal distribution function.Log-ratio and significance value (Student´s t-Test) calculation was performed as a basis for Volcanoplots.P-values were calculated using eBayes moderated t-statistics [5] .All proteins with a log-fold-change of >±1 were considered up-or downregulated between ∆fdxN or ∆fdxC strains (∆fdxN or ∆fdxC) and the WT (WT).All proteins with a 0.01 P-value (log10Adj.P-Value over 2.0) were considered significant.All strains were grown diazotrophically in RCV minimal medium under an N2 atmosphere (N2-fixing conditions).Boxes coloured in green are upregulated proteins, boxes in orange are downregulated proteins and boxes not coloured represent proteins that either are not up-or down-regulated or do not meet the 0.01 P-value cut-off.n/a stands for not applicable.S4.All strains carry an in-frame deletion of ∆nifD ∆modABC to ensure the expression of Fe-nitrogenase genes (anf).R. capsulatus strains were grown diazotrophically in RCV minimal medium under an N2 atmosphere.S4.All strains carry an in-frame deletion of ∆nifD ∆modABC to ensure the expression of Fe-nitrogenase genes (anf).R. capsulatus strains were grown diazotrophically in RCV minimal medium under an N2 atmosphere.All strains carry an in-frame deletion of ∆nifD ∆modABC to ensure the expression of Fe-nitrogenase genes (Anf).R. capsulatus strains were grown diazotrophically in RCV minimal medium under an N2 atmosphere.All proteins with a 0.01 P-value (log10Adj.P-Value over 2.0) were considered significant.All proteins with a log2-fold-change of value of >±1 were considered over or under produced, between ∆fdxN or ∆fdxC to WT. Coefficients of variation between 4 independent cultures provided in Table S4.and FdC (right, red) retrieved from UniProt [10,11] .Models are ribbon presentations of FdN and FdC with FeS clustercoordinating cysteines labelled and shown as stick models with sulphur in yellow.UniProt identifier codes and AlphaFold structure identifier codes are: for FdN: D5ARY6, AF-D5ARY6-F1 and for FdC: D5ARY7, AF-D5ARY7-F1.

Fig S1 .
Fig S1.Up-regulation of nitrogen fixation-related proteins in R. capsulatus ∆fdxN strain relative to R. capsulatus WT.Volcano plot displaying mean intensity log2-ratios of proteins (X-axis) versus significance values (-log10 adjusted P-values, Y-axis) between R. capsulatus ∆fdxN strains and R. capsulatus WT.N2 fixation-related proteins are shown in purple.All proteins with a log2-fold-change of >±1 were considered up-or down-regulated between R. capsulatus ∆fdxN and R. capsulatus WT strains.All proteins with a 0.01 P-value (log10Adj.P-Value over 2.0) were considered significant.Grey boxes highlight regions of log2fold values of -1> n <1 and log10P-values of <2.Coefficients of variation between 4 independent cultures provided in TableS4.All strains carry an in-frame deletion of ∆nifD ∆modABC to ensure the expression of Fe-nitrogenase genes (anf).R. capsulatus strains were grown diazotrophically in RCV minimal medium under an N2 atmosphere.

Fig. S2 .
Fig. S2.Up-regulation of nitrogen fixation-related proteins in R. capsulatus ∆fdxC strain relative to R. capsulatus WT.Volcano plot displaying mean intensity log2-ratios of proteins (X-axis) versus significance values (-log10 adjusted P-values, Y-axis) between R. capsulatus ∆fdxC strains and R. capsulatus WT.N2 fixation-related proteins are shown in purple.All proteins with a log2-fold-change of >±1 were considered up-or down-regulated between R. capsulatus ∆fdxC and R. capsulatus WT strains.All proteins with a 0.01 P-value (log10Adj.P-Value over 2.0) were considered significant.Grey boxes highlight regions of log2fold values of -1> n <1 and log10P-values of <2.Coefficients of variation between 4 independent cultures provided in TableS4.All strains carry an in-frame deletion of ∆nifD ∆modABC to ensure the expression of Fe-nitrogenase genes (anf).R. capsulatus strains were grown diazotrophically in RCV minimal medium under an N2 atmosphere.

Fig. S3 .
Fig. S3.No significant abundance changes in NifJ, HupA and HupB in ∆fdxN and ∆fdxC vs WT. (A) Scatter plot displaying mean intensity log2-ratios (X-axis) versus significance values (-log10 adjusted P-values, Y-axis) of NifJ, HupA and HupB in ∆fdxN compared to the WT.(B) Scatter plot displaying mean intensity log2-ratios (X-axis) versus significance values (-log10 adjusted P-values, Y-axis) of NifJ, HupA and HupB in ∆fdxC to compared to the WT.(A-B)All strains carry an in-frame deletion of ∆nifD ∆modABC to ensure the expression of Fe-nitrogenase genes (Anf).R. capsulatus strains were grown diazotrophically in RCV minimal medium under an N2 atmosphere.All proteins with a 0.01 P-value (log10Adj.P-Value over 2.0) were considered significant.All proteins with a log2-fold-change of value of >±1 were considered over or under produced, between ∆fdxN or ∆fdxC to WT. Coefficients of variation between 4 independent cultures provided in TableS4.

Fig. S5 .
Fig. S5.Unrooted phylogenetic tree of [Fe2S2]-cluster containing ferredoxins from R. capsulatus.R. capsulatus Fd sequences are coloured in green and numbered as according to the green key on the right.

Table S3 : Log2 ratios of N2 fixation proteins between R. capsulatus grown under N2-fixing conditions (N-free) and non-N2-fixing conditions (NH4 + ).
Boxes coloured in green are upregulated proteins, boxes in orange are downregulated proteins and boxes not coloured represent proteins that either are not up-or down-regulated or do not meet the 0.01 P-value cut-off.n/a stands for not applicable.